B. Walther, R. Pictet, J. David
Mar 25, 1974
Citations
1
Influential Citations
68
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Journal
The Journal of biological chemistry
Abstract
Abstract The thymidine analogue, 5-bromodeoxyuridine (BrdUrd) exerts a selective inhibition on cytodifferentiation of the embryonic rat pancreas. Pancreatic rudiments cultured in the presence of appropriate levels of BrdUrd exhibit essentially normal rates of protein, RNA, and DNA synthesis, but do not accumulate the specific acinar proteins; half-maximal inhibition of the synthesis and accumulation of chymotrypsin, amylase, ribonuclease, and procarboxypeptidases A and B occurs at levels of BrdUrd 50-fold below the levels required for half-maximal inhibition of the synthesis of total proteins. Tissues grown in the presence of these low levels of BrdUrd appear healthy from an ultrastructural point of view, but the presumptive acinar cells are devoid of either zymogen granules or large vesicular structures resembling zymogen granule membranes. However, many cells possess extensive rough endoplasmic reticulum. The enhanced ribosomal RNA synthesis observed in the normal differentiating pancreas is also observed in BrdUrd-treated pancreases. The possibility of a specific inhibition by BrdUrd of key metabolic pathways involving uridine, cytidine, or thymidine intermediates was tested. No significant effects on incorporation of precursors into phospholipids, sphingolipids, or glycoproteins were observed. There were also no observed effects of BrdUrd on acetate incorporation into sterols or free fatty acids. The possibility that degradation of BrdUrd via the thymidine or uridine catabolic pathways might produce deleterious metabolites was also tested. Attempts to reverse the BrdUrd effect by normal metabolites were unsuccessful. Furthermore, 5-bromouridine does not produce similar effects to BrdUrd. Among the many compounds tested, only thymidine effectively prevents the inhibitory action of BrdUrd. This inhibition is correlated with a decreased incorporation of BrdUrd into DNA. The fate of labeled BrdUrd in the pancreas cells was followed. The only low molecular weight compounds detected were free BrdUrd, and the mono-, di-, and triphosphates of 5-bromodeoxycytidine and BrdUrd. The great majority of the label rapidly accumulated in DNA. The kinetics of the BrdUrd inhibition, and its reversal suggest a mechanism of action involving a stable molecule such as DNA. The data collectively are consistent with a DNA-linked mechanism of BrdUrd inhibition. The inhibition is not the result of mutagenic action, since it is reversible and no altered proteins can be detected. A mechanism involving an altered binding of regulatory proteins to BrdUrd-containing DNA, and a consequent specific inhibition of the transcription of certain genes is proposed.